The present invention relates to a series of substituted 2-phenyl-4-quinazolinones compounds and substituted 2-phenyl4-alkoxy-quinazoline compounds; and in particular to their uses in treating human cancers and in inhibiting platelet aggregation.
Microtubules provide an important framework defining cellular morphology and are essential in the division and transport of cellular chromosomes. Consequently, the microtubule has become an important target for the design of new antimitotic anticancer agents. The antimitotic agents currently in clinical use include vinca alkaloids [Rowinsky, E. K.; Donehower, R. C. The clinical pharmacology and use of antimicrotubule agents in cancer chemotherapeutics. Pharmacol. Ther. 1992, 52, 35-84], which inhibit microtubule polymerization, and taxoids, which promote microtubule assembly [Verweij, J.; Clavel, M.; Chevalier, B. Paclitaxel (Taxol) and docetaxel (Taxotere): not simply two of a kind. Ann. Oncol. 1994, 5, 495-505]. Colchicine is another well-known antimitotic agent; however, being too toxic to be used as anticancer agent, it is used clinically only as an antigout agent [Hastie, S. B. Interactions of colchicine with tubulin. Pharmacol. Ther. 1991, 51, 377-401; Brossi,A; Yeh, H. J.; Chrzanowska, M.; Wolff, J.; Hamel, E.; Lin, C. M.; Quinn, F.; Suffness, M.; Silverton, J. Colchicine and its analogues: recent findings. Med. Res. Rev 1988, 8, 77-94].
In recent years, some of the inventors of the present application and their co-workers have designed and synthesized three type of heterocyclic ketones, the 2-phenyl4-qinolones (PQ), 2,3-dihydro-2-phenyl-4-quinolones (DHPQ) and 2-phenyl-1,8-naphthyridin4-ones (PN) as novel antimitotic agents and have established a preliminary structure-activity relationships [Brossi, A.; Yeh, H. J.; Chrzanowska, M.; Wolff, J.; Hamel, E.; Lin, C. M.; Quinn, F.; Suffness, M.; Silverton, J. Colchicine and its analogues: recent findings. Med. Res. Rev. 1988, 8, 77-94; Kuo, S. C.; Lee, H. Z.; Juang, J. P.; Lin, Y. T.; Wu, T. S.; Chang, J. J.; Ledniced, D.; Paull, K. D.; Lin, C. M.; Hamel, E.; Lee, K. H. Synthesis and cytotoxicity of 1,6,7,8 and 4xe2x80x2-substituted 2-phenyl-4-quinolones and related compounds: identification as antimitotic agents interacting with tubulin. J. Med. Chem. 1993, 36, 1146-1156; Li, L.; Eang, H. K.; Kuo, S. C.; Lednicer, D.; Lin, C. M.; Hamel, E.; Lee, K. H. 2xe2x80x2,3xe2x80x2,4xe2x80x2,5xe2x80x2,5,6,7-Substituted 2-phenyl-4-quinolones and related compounds: their synthesis, cytotoxicity, and inhibition of tubulin polymerization. J. Med. Chem. 1994, 37 (8),1126-1135; Li, L.; Wang, H. K.; Kuo, S. C.; Wu, T. S.; Mauger, A.; Lin, C. M.; Hamel, E.; Lee, K. H. Synthesis and biological evaluation of 3xe2x80x2,6xe2x80x2,7-substituted 2-phenyl4-quinolones as antimitotic antitumor agents. J. Med. Chem. 1994, 37 (20), 3400-3407;Xia, Y.; Yang, Z. Y.; Xia, P.; Bastow, K. F.; Tachibana, Y.; Kuo, S. C.; Hamel, E.; Hackl, T.; Lee, K. H. Synthesis and biological evaluation of 6,7,2xe2x80x2,3xe2x80x2,4xe2x80x2-substituted-1,2,3,4-tetrahydro-2-phenyl-4-quinolones as a new class of antimitotic agents. J. Med. Chem. 1998, 41 (7), 1155-1162; Xia, Y; Yang, Z. Y.; Xia, P.; Bastow, K. F.; Tachibana, Y.; Kuo, S. C.; Hamel, E.; Hackl, T.; Lee, K. H. Synthesis and biological evaluation of 6,7,2xe2x80x2,3xe2x80x2,4xe2x80x2-substituted-1,2,3,4-tetrahydro-2-phenyl-4-quinolones as a new class of antimitotic agents. J. Med. Chem. 1998, 41 (7), 1155-1162; Chen, K.; Kuo, S. C.; Hsih, M. C.; Mauger, A.; Lin, C. M.; Hamel, E.; Lee, K. H. 2xe2x80x2,3xe2x80x2,4xe2x80x2,5,6,7-Substituted 2-phenyl-1,8-naphthyridin-4-ones: their synthesis, cytotoxicity, and inhibition of tubulin polymerization. J. Med. Chem. 1996, 40 (14), 2266-2275; Chen, K.; Kuo, S. C.; Hsih, M. C.; Mauger, A.; Lin, C. M.; Hamel, E.; Lee, K. H. Synthesis and biological evaluation of substituted 2-aryl-1,8-naphthyridin-4(1H)-ones as antimitotic antitumor agents that inhibit tubulin polymerization. J. Med. Chem. 1997, 40 (19), 3049-3056.]. The structures of PQ, DHPQ and PN are shown as follows: 
Among these three types of heterocyclic ketones, the common structural feature is a biaryl system composed of A- and C-rings that are linked by an interposed B-ring or sometimes by a hydrocarbon bridge. However, some minor structural differences also exist.
In the PQ system, when functional groups with nonbonding electrons, e.g. xe2x80x94OCH3, xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94, xe2x80x94NRRxe2x80x2, Cl and F (PQ1-6), were placed at the 6-position of the A-ring and the 3xe2x80x2-position of the C-ring, activity was very potent. These two functional groups are about 10 to 11 xc3x85 apart, and these two groups possibly may interact with the tubulin binding domain by acting as H-receptors. Thus, they might contribute significantly to the potency of PQ compounds. In the DHPQ system, the 6- and 3xe2x80x2-substituents (DHPQ1) also plays a decisive role in activity. However, in the PN system, when the 3xe2x80x2-substituent is fixed (e.g. OCH3), the identity of the 6-substituent e.g. H (PN-1), CH3 (PN-2) or Cl (PN-3), does not noticeably affect activity. This finding is unique to the PN system and differentiates it from the PQ and DHPQ systems. The structures of PQI to PQ6, PN-1 to PN-3 and DHPQ1 are shown in the followings: 
The antitumor activities of 2,3-dihydro-2-aryl-4-quinazolinones (DHPQZ) were reported around 1970 [Yale, H. J.; Kalkstein, M. Substituted 2,3-dihydro-4(1H)-quinazolinones. A new class of inhibitors of cell multiplication. J. Med. Chem. 1967, 10, 334-336; Neil, G. L.; Li, L. H.; Buskirk, H. H.; Moxicy, T. E. Antitumor effects of the antisperrnatogenic agent, 2,3-dihydro-2-(1-naphthyl)-4(1H)-quinazolinones. Cancer Chemother. 1972,56,163-173]. Amore recent reevaluation of this type of compounds by NCl against human tumor cell lines reconfirmed that, like colchicine, they are effective inhibitors of tubulin polymerization [Hamel, E.; Lin, C. M.; Plowman, J.; Wang, H. K.; Lee, K. H.; Paull, K. D. Antitumor 2,3-dihydro-2-(aryl)-4(1H)-quinazolinone derivatives. Interactions with tubulin. Biochem. Pharmacol. 1996, 51, 53-59]. At the same time, 2-styrylquinazolin-4-ones (SQZ) were also identified as potent inhibitors of tubulin polymerization [Jiang, J. B.; Hesson, D. P.; Dusak, B. A.; Dexter, D. L.; Kang, G. J.; Hamel, E. Synthesis and biological evaluation of 2-styryl-quinazolin-4(3H)-ones, a new class of antimitotic anticancer agents which inhibit tubulin polymerization. J. Med. Chem. 1990, 33, 1721-1728; Lin, C. M.; Kang, G. J.; Roach, M. C.; Jiang, J. B.; Hesson, D. P.; Luduena, R. F.; Hamel, E. Investigation of the mechanism of the interaction of tubulin with derivatives of 2-styrylquinazolin-4(3H)-one. Mol. Pharmacol. 1991, 40, 827-832.]. DHPQZ and SQZ compounds have the following structures: 
The present invention synthesizes compounds having the structures of the following formulas (I) to (IV): 
wherein
R2xe2x80x2, R3xe2x80x2, R4xe2x80x2, and R5xe2x80x2 independently are H, (CH2)nCH3, OH, O(CH2)nCH3, X, or NR8R9, wherein n is an integer of 0xcx9c4, X is F, Cl, or Br, and R8 and R9 independently are H or (CH2)nCH3, wherein n is defined as above;
R is (CH2)nCH3 or (CH2)nCOO(CH2)nCH3, wherein n is defined as above; and
R6 and R7 independently are H, (CH2)nCH3, OH, O(CH2)nCH3, X, NR8R9, 
or R6 and R7 together is xe2x80x94OCH2Oxe2x80x94, wherein n, X, R8 and R9 are defined as above.
The compounds (I) and (II) were evaluated for cytotoxicity and as inhibitors of tubulin polymerization in the present invention. Some of them show potent cytotoxicity against a panel of human tumor cell lines and show potent inhibition of tubulin polymerization, and thus have great potential to be used as a therapeutically effective component in a pharmaceutical composition for treating cancer.
The compounds (III) and (IV) were found potent in inhibiting aggregation of platelet in the present invention, and thus are useful as a therapeutically effective component in a pharmaceutical composition for inhibiting aggregation of platelet.
In one aspect of the present invention a series of 6,7,2xe2x80x2,3xe2x80x2,4xe2x80x2,5xe2x80x2-substituted 2-phenyl-4-quinazolones having the formula (I) are synthesized: 
wherein R2xe2x80x2, R3xe2x80x2, R4xe2x80x2, R5xe2x80x2, R6 and R7 are defined as above.
Preferably, R2xe2x80x2, R3xe2x80x2, R4xe2x80x2 and R5xe2x80x2 in the formula (I) independently are H or O(CH2)nCH3, and at least one of R2xe2x80x2, R3xe2x80x2, R4xe2x80x2 and R5xe2x80x2 is O(CH2)nCH3, wherein n is defined as above. More preferably, R2xe2x80x2, R4xe2x80x2 and R5xe2x80x2 are H, and R3xe2x80x2 is methoxy.
Preferably, R6 and R7 in the formula (I) independently are H, O(CH2)nCH3, X, NR8R9, 
or R6 and R7 together is xe2x80x94OCH2Oxe2x80x94, wherein n, X, R8 and R9 are defined as in claim 1. More preferably, R6 and R7 independently are H, O(CH2)nCH3, X, NR8R9, 
or R6 and R7 together is xe2x80x94OCH2Oxe2x80x94, wherein n, X, R8 and R9 are defined as above. Most preferably, R7 is H, and R6 is NR8R9, 
wherein R8 and R9 are defined as above, and preferably, R8 and R9 are methyl
Preferably, R6 and R7 in the formula (I) independently are H, methoxy or X, wherein X is defined as above.
In another aspect, the present invention provides a series of 6,7,2xe2x80x2,3xe2x80x2,4xe2x80x2,5xe2x80x2-substituted 2,3-dihydro-2-phenyl4-quinazolones having the following formula (II): 
wherein R2xe2x80x2, R3xe2x80x2, R4xe2x80x2, R5xe2x80x2, R6 and R7 are defined as above.
Preferably, R7 in the formula (II) is H.
Preferably, R2xe2x80x2, R3xe2x80x2, R4xe2x80x2 and R5xe2x80x2 in the formula (II) independently are H or O(CH2)nCH3, provided that at least one of them is O(CH2)nCH3, wherein n is defined as above. More preferably, R2xe2x80x2, R4xe2x80x2 and R5xe2x80x2 are H, and R3xe2x80x2 is methoxy.
Preferably, R6 in the formula (II) is X, wherein X is defined as above. More preferably, wherein X is Cl.
In further another aspect, the present invention provides a series of 3,6,7,2xe2x80x2,3xe2x80x2,4xe2x80x2,5xe2x80x2-substituted 2-phenyl-4-quinazolones having the following formula (III) and a series of 6,7,2xe2x80x2,3xe2x80x2,4xe2x80x2,5xe2x80x2-substituted 2-phenyl-4-alkoxy-quinazolines (IV): 
wherein R2xe2x80x2, R3xe2x80x2, R4xe2x80x2, R5xe2x80x2, R, R6 and R7 are defined as above.
Preferably, R5xe2x80x2, R6 and R7 in the formulas (III) and (IV) are all H.
Preferably, n in the formulas (III) and (IV) is 0 or 1.
Preferably, R in the formulas (III) and (IV) is (CH2)nCH3, wherein n is 0 or 1, and preferably n is 1.
Preferably, R2xe2x80x2, R3xe2x80x2, and R4xe2x80x2 in the formulas (III) and (IV) independently are H or OCH3. More preferably, R2xe2x80x2, R3xe2x80x2, and R4xe2x80x2 are all H. Alternatively, R2xe2x80x2 and R3xe2x80x2 are both H, and R4xe2x80x2 is OCH3.
The present invention also discloses a pharmaceutical composition for the treatment of cancer, which comprises a therapeutically effective amount of a compound of the formula (I) or (II) or a pharmaceutically acceptable salt thereof, as an active ingredient, in admixture with a pharmaceutically acceptable carrier or diluent for the active ingredient.
The present invention also provides a method of treating a cancer comprising administering a therapeutically effective amount of a compound of the formula (I) or (II) to a subject suffering from cancer.
The present invention also discloses a pharmaceutical composition for the inhibition of aggregation of platelet, which comprises a therapeutically effective amount of a compound of the formula (III) or (IV), preferably the formula (III), or a pharmaceutically acceptable salt thereof, as an active ingredient, in admixture with a pharmaceutically acceptable carrier or diluent for the active ingredient.
The present invention also provides a method of inhibiting an aggregation of platelet in a patient comprising administering a therapeutically effective amount of a compound of the formula (III) or (IV), preferably the formula (III), to said patient.
The starting 4,5-substituted 2-aminobenzamides (3, 10-11, 16, 25-29), needed for the synthesis of our target compounds, were prepared according to Schemes 1-4. As shown in Scheme 1, 2-amino-5-fluoro-benzamide (3) was prepared by treating commercially available 2-amino-5-fluoro-benzoic acid (1) first with SOCl2, then with ammonia. In Scheme 2, the 4-methoxy and 4,5-dimethoxy-2-aminobenzaimdes (10, 11) were prepared by converting the xe2x80x94COOH group of the starting 2-nitrobenzoic acids (4, 5) to xe2x80x94CONH2 (8, 9) followed by reduction of the 2-NO2 group to 2-NH2 (10, 11). In the same manner, 4,5-methylenedioxy-2-aminobenzamide (16) was obtained from 4,5-methylenedioxy-2-nitrobenzoic acid (13) after oxidation of the xe2x80x94CHO group of 4,5-methylenedioxy-2-nitrobenzaldehyde (12) (Scheme 3). Finally, according to Scheme 4, 6-alkylamino-2-aminobenzamides (25-29) were prepared by first converting the xe2x80x94COOH group of 5-chloro-2-nitrobenzoic acid (17) to xe2x80x94CONH2 (19) followed by nucleophilic displacement of the 5-chloro group by different secondary amines, and subsequent hydrogenation.
As illustrated in Scheme 5, target compounds 41-59 were prepared by reacting the above starting materials (3, 10-11, 16, 25-29, respectively) with methoxybenzaldehydes (33-40) in N,N-dimethylacetamide (DMAC) in the presence of NaHSO3. Thermal cyclodehydration/dehydrogenation resulted in substituted 2-phenyl-4-quinazolinones (41-59) in high yields. 
The synthesis of 2,3-dihydro-2-phenyl-4-quinazolinones (60-68) is described in Scheme 6. The preparation of 2,3-dihydro-3xe2x80x2-methoxy-2-phenyl-4-quinazolinone (61) is detailed below as an example.
First, a mixture of 2-aminobenzamide (30) and 3-methoxybenzaldehyde (34) in DMAc was heated to 80xc2x0 C. for 1 hr. Subsequent purification with column chromatography afforded 61 (mp 148-150xc2x0 C. ) and 43 (mp 177-179xc2x0 C.) in yields of 75% and 15%, respectively. The chemical structure of 43 was confirmed by IR, NMR and MS spectral analysis as 3xe2x80x2-methoxy-2-phenyl-4-quinazolinone. For 61, the molecular formula was determined by elemental and MS analysis (m/z 254, M+) as C15H14N2O2, which matches the expected 2,3-dihydro-3xe2x80x2-methoxy-2-phenyl-4-quinazolinone. The 1H NMR of 61 showed three additional proton signals not seen in the spectrum of 43: 5.73 (H-2), 7.15 (N1xe2x80x94H) and 8.34 (N3xe2x80x94H). The 2D HMBC spectrum of 61 showed the expected long range coupling between H-2 and C-4 (xcex4163.77), C-8a (xcex4147.98), C-2xe2x80x2 (xcex4119.11) and C-6xe2x80x2 (xcex4112.75). All spectral data confirmed our assignment of 61 as 2,3-dihydro-3xe2x80x2-methoxy-2-phenyl-4-quinazolinone.
In order to increase the yield of 61, reaction conditions were adjusted to minimize the unwanted dehydrogenation of 61 and convertism to 43. First, we lowered the reaction temperature to 25xc2x12xc2x0 C. and extended the reaction time to 4 hr. However, these conditions only resulted in a lower conversion as indicated by the considerable amount of starting materials (30, 34) detected by TLC in the reaction mixture. Next, we again carried out the reaction, at 25xc2x12xc2x0 C. in DMAC, but also incorporated a catalytic amount of p-toluenesulfonic acid. The reaction was completed in 30 min and the subsequent work up resulted in high yield (89.0%) of 61 and minor yield (4.0%) of 43. Consequently, the same reaction condition was adopted in the subsequent preparation of 60 and 62-68, and resulted in high yields of all desired products.
Thus, all 2,3-dihydro products (60-68) have been prepared as racemic mixtures. Resolution of these racemic mixtures has not been attempted in this work, but will be pursued if unusual biological activity is found in our forthcoming studies.
We also used various benzamides (10-11, 16, 25-29) with electron donating groups (e.g. xe2x80x94OCH3, xe2x80x94OCH2Oxe2x80x94, xe2x80x94NRRxe2x80x2) on the benzene ring as starting materials. Unexpectedly, instead of obtaining our desired 2,3-dihydro products, the corresponding dehydrogenated products (52-59) were produced in high yields. TLC check analysis of the crude products did show materials resembling the expected 2,3-dihydro derivatives. However, they were readily converted to their corresponding dehydrogenated derivatives (52-59) via spontaneous dehydrogenation during the work up procedures, such as column chromatography or recrystallization.
Finally, we used 5-fluorobenzamides (3) as starting material in the attempted preparation of 2,3-dihydro-6-fluoro-2-phenyl-4-quinazolinone. However, although F and Cl belong to the same class of electron withdrawing group, the fluorinated 2,3-dihydro derivative was much less stable than the Cl-containing counterpart (68) and readily underwent spontaneous dehydrogenation to give 51 during the final work up.
Summarizing the above results, we concluded that 2,3-dihydro-2-phenyl-4-quinazolinones containing electron donating groups or fluorine at the 6 or 7 position easily underwent spontaneous dehydrogenation. Thus, preparing these compounds for cytotoxic evaluation would be impractical, as they are readily undergo spontaneous dehydrogenation and would not be stable in the solvent required for biological testing.
The synthetic pathway for target compounds, 69-76, and 77-81 series, were illustrated in Scheme 7, the synthesis of two representing samples (69, 77) was detailed below.
The starting material, 2-phenyl-4-quinazolinone (41) was treated with NaH in THF, followed by methylation with methyl iodide to give 69 (mp 50-51xc2x0 C.) and 77 (mp 125-127xc2x0 C. ) in the yield ratio of 1:2.2. From the elemental analysis and mass spectral data (m/z 236), their molecular formulae were both determined as C15H12N2O. This result led to our assumption that they might be O-methyl and N-methyl isomers, such assumption was proven to be supported by their 1H-NMR spectral evidence, in which the signal of methyl group for compound 69 at xcex44.26 was assigned to OCH3 protons. In contrast, the signal of methyl group for compound 77 at xcex43.47 was attributed to its N-methyl protons. Furthermore, we compared their HMBC spectra of 2D NMR and found that the O-methyl protons of 69 exhibited long range coupling only with C-4 (xcex4167.00), whereas the N3-methyl protons of 77 showed long range coupling with both C-2 (xcex4156.10) and C-4 (xcex4162.76). Based on the above spectral evidence, we confirmed 69 as 4-methoxy-2-phenylquinazoline and 77 as N3-methyl-2-phenyl-4-quinazolinone.
Following similar synthetic procedures for 69 and 77, compound 41 was subjected to alkylation by ethyl iodide and ethyl bromoacetate respectively, to yield primarily O-ethyl (70) and O-ethoxycarbonylmethyl (71) derivatives respectively. Some minor yield of N3-ethyl (78) and N3-ethoxycarbonylmethyl derivatives (79), respectively, was also found.
Next, when ethyl 4-chlorobutyrate and ethyl 5-bromovalerate were used separately in the alkylation of compound 41, only their corresponding O-alkylated derivatives (72, 73) were produced, and no trace of N-alkylated products were detectable by TLC.
Again, when using 2xe2x80x2,3xe2x80x2,4xe2x80x2-substituted 2-phenyl-4-quinazolinones (42-44) as the starting materials, their ethylation products were found to be mainly, their corresponding O-ethyl derivatives (74-76), although small quantity of their corresponding N-ethyl products (80-81) were also present. 